The recent breakthrough in hypertension research reveals that a novel WNK kinase family [(with-no- lysine (K)] and its two key down-stream substrates SPAK (Ste20/SPS1-related proline/alanine-rich kinase) and its homolog OSR1 (oxidative stress-responsive kinase 1) are evolutionarily conserved regulators of ion transporters by altering their net phosphorylation state and play an important role in renal salt handling and in the pathogenesis of hypertension. Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) transports 1Na+, 1K+, and 2Cl- ions into cells and is important in regulation of intracellular Na+ and Cl-, cell volume, and K+ uptake in the central nervous system (CNS) under physiological conditions. Previous studies from our lab and others' clearly indicate that over-stimulation of NKCC1 activity contributes to cerebral ischemic damage. Under ischemic conditions, NKCC1 activation causes intracellular Na+ and Cl- overload in astrocytes and neurons. The intracellular Na+ overload subsequently stimulates the reverse mode operation of Na+/Ca2+ exchange and leads to a delayed, secondary cytosolic Ca2+ rise and Ca2+ dysregulation in ER and mitochondria. Most importantly, either pharmacological inhibition or genetic ablation of NKCC1 shows significant neuroprotective effects in in vivo focal ischemia model (middle cerebral artery occlusion, MCAO) and in vitro ischemia model. Despite of the neuroprotective effects in ischemic brain damage by blocking NKCC1 activity, it remains unknown how NKCC1 protein is stimulated in ischemic brains and what are the up-stream regulatory mechanisms. Our pilot study revealed robust stimulation of the WNK-SPAK/OSR1 signaling pathway in ischemic brains. The goal of this project is to investigate whether the cerebral WNK3-SPAK-NKCC1 signaling pathway contributes to ischemic brain damage and whether blocking the WNK3/SPAK kinases with a newly discovered WNK-SPAK pathway inhibitor STOCK1S-50699 or transgenic knockout of WNK3 (WNK3 KO), or SPAK (SPAK KO) is neuroprotective. Our preliminary study shows that WNK3 KO mice exhibited significantly reduced infarct volume, less axonal demyelination, and accelerated neurobehavioral recovery. New data in this resubmission illustrates a ~70% reduction in infarct volume in SPAK KO mice. These data provide fresh insight into the role of ion transporters and their regulatory kinases in ischemic neuroglial injury. Completion of this project will help us to determine whether the WNK3-SPAK kinase complex presents a compelling target for novel neuroprotective strategies for ischemic brain injury. Our study will pave a foundation for developing new WNK-SPAK inhibitors for ischemic brain damage therapy.